Method of nucleic acid sequence selection

ABSTRACT

The present invention provides a method for the rapid isolation and recovery of a desired target DNA or RNA molecules from a mixture or library containing such molecules. The method involves the use of biotinylated probes and enzymatic repair-cleavage to eliminate undesired library members from a sample.

FIELD OF THE INVENTION

[0001] The invention relates to an improved method for isolating andrecovering target DNA or RNA molecules having a desired nucleotidesequence. Specifically, it relates to a method for the rapid isolationof specific nucleic acid target molecules.

BACKGROUND OF THE INVENTION

[0002] The ability to clone gene sequences has permitted inquiries intothe structure and function of nucleic acids, and has resulted in anability to express highly desired proteins, such as hormones, enzymes,receptors, antibodies, etc., in diverse hosts.

[0003] The most commonly used methods for cloning a gene sequenceinvolve the in vitro use of site-specific restriction endonucleases, andligases. In brief, these methods rely upon the capacity of the“restriction endonucleases” to cleave double-stranded DNA in a mannerthat produces termini whose structure (i.e. 3′ overhang, 5′ overhang, orblunt end) and sequence are both well defined. Any such DNA molecule canthen be joined to a suitably cleaved vector molecule (i.e. a nucleicacid molecule, typically double-stranded DNA, having specializedsequences which permit it to be replicated in a suitable host cell)through the action of a DNA ligase. The gene sequence may then beduplicated indefinitely by propagating the vector in a suitable host.Methods for performing such manipulations are well-known (see, forexample, Perbal, B. A Practical Guide to Molecular Cloning, John Wiley &Sons, NY, (1984), pp. 208-216; Maniatis, T., et al. (In: MolecularCloning. A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1982); Old, R. W. et al., In: Principles of GeneManipulation, 2^(nd) Ed., University of California Press, Los Angeles,(1981), all herein incorporated by reference).

[0004] In some cases, a gene sequence of interest is so abundant in asource that it can be cloned directly without prior purification orenrichment. In most cases, however, the relative abundance of a desiredtarget DNA molecule will require the use of ancillary screeningtechniques in order to identify the desired molecule and isolate it fromother molecules of the source material.

[0005] A primary screening technique involves identifying the desiredclone based upon its DNA sequence via hybridization with a nucleic acidprobe. In situ filter hybridization methods are particularly well known(see, Sambrook, J., et al., In: Molecular Cloning, A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)). In such methods, bacteria are lysed on the surface of themembrane filter and then incubated in the presence of a detectablylabelled nucleic acid molecule whose sequence is complimentary to thatof the desired sequence. If the extract contains the desired sequence,hybridization occurs and thereby binds the labelled molecule to theadsorbent surface. The detection of the label on the adsorbent surfacereveals that the bacteria sampled contained the desired cloned sequence.

[0006] Although these screening methods are useful and reliable, theyrequire labor-intensive and time consuming steps such as filterpreparation and multiple rounds of filter hybridization and colonyplatings/phage infections. Generally, these procedures will screen up to10⁶ colonies effectively, but may take weeks or months to yield thedesired clone.

[0007] Recently, other approaches have been developed to isolaterecombinant molecules which have eliminated the tedious filter-handlingprocedure. These approaches employ conventional hybridization technologycoupled with chromatography or magnetic particle technology. Rigas, B.et al., for example, reported a method for isolating one plasmid speciesfrom a mixture of two plasmid species. In the disclosed method, circulardouble-stranded plasmid DNA is hybridized to a RecA protein-coatedbiotinylated probe to form a stable triple-stranded complex, which isthen selectively bound to an agarose-streptavidin column (Rigas, B. etal., Proc. Natl. Acad. Sci. (U.S.A.) 83: 9591-9595 (1986)). The methodthus permits the isolation of cloned double-stranded molecules withoutrequiring any separation of the strands.

[0008] A DNA isolation method, termed “triplex affinity capture,” hasbeen described in which a specific double-stranded genomic DNA ishybridized to a biotinylated homopyrimidine oligonucleotide probe toform a “triplex complex,” which can then be selectively bound tostreptavidin-coated magnetic beads (Ito, T. et al., Nucleic Acids Res.20: 3524 (1992); Ito, T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:495-498 (1992)). Takabatake, T. et al. have described a variation ofthis technique that employs a biotinylated purine-rich oligonucleotideprobe to detect and recover the desired nucleic acid molecule(Takabatake, T. et al., Nucleic Acid Res, 20: 5853-5854 (1992)). Apractical drawback with these particular approaches is that they arerestricted to isolation of target DNA sequences containinghomopurine-homopyrimidine tracts.

[0009] Fry, G. et al. discuss a method for sequencing isolated M13-LacZphagemids (Fry, G. et al., BioTechniques 13:124-131 (1992)). In thismethod, a clone is selected and the phagemid DNA is permitted tohybridize to a biotinylated probe whose sequence is complementary to thephagemid's lacZ region. The biotinylated probe is attached to astreptavidin-coated paramagnetic bead. Since the DNA bound to the beadcan be separated from unbound DNA, the method provides a means forseparating the cloned sequence from the bacterial sequences that areinevitably present (Fry, G. et al., BioTechniques 13: 124-131 (1992)).

[0010] Still another method of screening recombinant nucleic acidmolecules is described by Kwok, P. Y. et al. This method, which is anextension of PCR-based screening procedures uses an ELISA-basedoligonucleotide-ligation assay (OLA) to detect the PCR products thatcontain the target source (Kwok, P. Y. et al., Genomics 13: 935-941(1992)). The OLA employs an “reporter probe” and aphosphorylated/biotinylated “anchor” probe, which is captured withimmobilized streptavidin (Landegren, U. et al., Science 241:1077-1080(1988)).

[0011] The isolation of target DNA from a complex population using asubtractive hybridization technique has also been described (Lamar, E.E. et al., Cell 37:171-177 (1984); Rubenstein, J. L. R. et al., NucleicAcids Res. 1:4833-4842 (1990); Hedrik, S. M. et al., Science 308:149-153(1984); Duguid, J. R. et al., Proc. Natl. Acad. Sci. (U.S.A.)85:5738-5742 (1988)). In such “subtractive hybridization” screeningmethods, the cDNA molecules created from a first population of cells ishybridized to cDNA or RNA of a second population of cells in order to“subtract out” those cDNA molecules that are complementary to nucleicacid molecules present in the second population and thus reflect nucleicacid molecules present in both populations

[0012] The method is illustrated by Duguid, J. R. et al. (Proc. Natl.Acad. Sci. (U.S.A.) 85:5738-5742 (1988)) who used subtractivehybridization to identify gene sequences that were expressed in braintissue as a result of scrapie infection. A cDNA preparation made fromuninfected cells was biotinylated and permitted to hybridize with cDNAmade from infected cells in a sample. Sequences in common hybridized toone another, and were removed from the sample through the use of abiotin-binding (avidin) resin.

[0013] Weiland, I. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 87:2720-2724(1990)) reported an improved method of subtractive hybridization inwhich tester DNA was cleaved with a restriction endonuclease, and thenpermitted to hybridize to sheared driver DNA at high C₀t values (“C₀t”is the product of the initial concentration of DNA and the time ofincubation). By cloning the double-stranded, PCR-amplified, unique DNAmolecules into a plasmid vector, it was possible to obtain an enrichmentin the relative proportion of target sequences recovered.

[0014] Rubenstein, J. L. R. et al. (Nucleic Acids Res. 18:4833-4842(1990)) reported a further improvement in subtractive hybridizationmethods that employed single-stranded phagemid vectors to provide boththe target and tester DNA. In the method, hybridized phagemidDNA-biotinylated driver strand complexes are separated from unhybridizedDNA by the addition of streptavidin. Unhybridized single-stranded DNAwas subsequently converted to the double-stranded form using Taq DNApolymerase and an oligonucleotide complementary to a common region foundwithin the single-stranded DNA. The use of this method is, however,limited by the need to follow a rigorous single-stranded phagemidpurification protocol in order to obtain a preparation virtually free ofcontaminant double-stranded DNA. (Rubenstein, J. L. R. et al., NucleicAcids Res. 18: 4833-4841 (1990)).

[0015] In sum, methods for isolating particular target nucleic acidmolecules are restricted by the abundance of the DNA target sequence,and by time-consuming steps. Accordingly, a method that would expeditethe isolation of desired target nucleic acid molecules and that couldyield essentially pure target DNA would be highly desirable.

SUMMARY OF THE INVENTION

[0016] The present invention provides a method for rapidly isolatingnucleic acid molecules having a desired nucleotide sequence from otherundesired-nucleic acid molecules. Significantly, the present inventionfurther relates to an improved method of screening target nucleic acidmolecules employing hybridization methodology combined with ligandseparation, DNA repair, and restriction enzyme digestion technology.

[0017] In detail, the invention provides a method for recovering adesired target nucleic acid molecule from a sample containing a mixtureor library of single-stranded nucleic acid containing the molecule,wherein the method comprises the steps:

[0018] A. incubating the sample containing the nucleic acid mixture orlibrary in the presence of a primer nucleic acid molecule complementaryto a sequence of the desired target molecule; the incubation being underconditions sufficient to permit the template-dependent extension of theprimer to thereby generate a double-stranded desired target molecule;

[0019] B. transforming single-stranded and double-stranded members ofthe mixture or library into a host cell, and

[0020] C. recovering the desired molecule from the cell.

[0021] The invention additionally provides the embodiment wherein priorto commencing step A, the method comprises the presteps:

[0022] (1) incubating an initial sample containing the nucleic acidmixture or library in the presence of a haptenylated nucleic acid probemolecule, the probe molecules having a sequence complimentary to anucleotide sequence of the desired target molecule; the incubation beingunder conditions sufficient to permit the probe to hybridize to thedesired target molecule and to thereby generate a hybridized moleculewherein the target molecule is bound to the probe;

[0023] (2) incubating the sample containing the nucleic acid mixture orlibrary and biotinylated probe-target hybridized molecules of prestep(1) in the presence of a binding ligand of the hapten of thehaptenylated probe, the binding ligand being conjugated to support; theincubation being sufficient to permit the probe molecules, and theprobe-target hybridized molecule to become bound to the binding ligandof the support;

[0024] (3) recovering the probe-target hybridized molecules bound to thesupport from the nucleic acid mixture or library and any unboundbiotinylated probe-target hybridized molecules of prestep (2); and

[0025] (4) incubating the recovered support containing the boundprobe-target hybridized molecules under conditions sufficient toseparate the strands of double-stranded molecules; the incubationthereby releasing the hybridized target molecule from the biotinylatedprobe, and generating a sample single-stranded desired target moleculefor use in step (A).

[0026] The invention additionally provides the embodiment wherein thesingle-stranded nucleic acid molecule of the sample contains anucleotide analog, and wherein after completing step A, but prior tocommencing step B, the method additionally comprises the presteps:

[0027] (1′) incubating the generated double-stranded molecules in thepresence of a nuclease capable of degrading nucleic acid containingnucleotide analog residues; and

[0028] (2′) incubating non-degraded nucleic acid with a primer underconditions sufficient to permit the primer to be extended in atemplate-dependent manner.

[0029] The invention additionally provides the embodiment wherein instep A, the template-dependent extension of the primer is conducted inthe presence of a nuclease resistant nucleotide analog to therebygenerate a double-stranded desired target molecule containing a residueof the nucleotide analog; and wherein prior to commencing the step B,the method additionally comprises the presteps:

[0030] (1″) incubating the generated double-stranded desired targetmolecule in the presence of a nuclease, wherein the nuclease issubstantially unable to cleave a nucleic acid molecule containing thenucleotide analog residue, but is substantially capable of degradingboth single-stranded nucleic acid molecules and double-stranded nucleicacid molecules that lack the nucleic acid analog residue; the incubationbeing under conditions sufficient to permit such degradation, andthereby substantially eliminating both single-stranded nucleic acidmolecules and double-stranded nucleic acid molecules that lack thenucleic acid analog residue from the sample; and thereby forming apreparation having a substantial enrichment of the desired targetmolecule relative to the initial sample; and

[0031] (2″) recovering the desired molecule from the preparation ofprestep (1″) to thereby form a library or mixture for the step B. Theinvention particularly concerns the embodiments of the above methodswherein the desired target nucleic acid molecule is a DNA molecule, asingle-stranded nucleic acid molecule, a circular nucleic acid moleculeand most preferably, a single-stranded, circular DNA molecule.

[0032] The invention particularly concerns the embodiments of the abovemethods wherein the desired target nucleic acid molecule is a DNA or RNAmolecule, a single-stranded nucleic acid molecule, a circular nucleicacid molecule and most preferably, a single-stranded, circular DNAmolecule.

[0033] The invention also concerns the embodiments of the above methodswherein the hapten is biotin, and wherein the binding ligand of thehapten is avidin, streptavidin, or antibody or antibody fragments thatbind biotin.

[0034] The invention also concerns the embodiments of the above methodswherein the support is a bead, especially a paramagnetic bead that canbe recovered by magnetic means or other physical separation.

[0035] The invention also concerns the embodiments of the above methodswherein the sequence of the primer molecule may be complementary to thesame sequence of the desired target molecule as the probe molecule, orit may be complementary to a different sequence.

BRIEF DESCRIPTION OF THE FIGURE

[0036]FIG. 1 provides a diagrammatic illustration of a preferredembodiment of the isolation method of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention concerns an improved method for rapidlyisolating a “desired” nucleic acid “clone” from a mixture or library ofcloned molecules. The “clones” of the present invention comprisecircular or linear DNA or RNA molecules that may be eithersingle-stranded or double-stranded. Typically, such clones or librarieswill comprise plasmids or other vectors (such as viral vectors) thathave been engineered to contain a fragment of DNA or RNA derived from asource such as a homogeneous specimen (such as cells in tissue culture,cells of the same tissue, etc.), or a heterogeneous specimen (such as amixture of pathogen-free and pathogen-infected cells, a mixture of cellsof different tissues, species, or cells of the same or different tissueat different temporal or developmental stages, etc.). The cells, if any,of these nucleic acid sources may be either prokaryotic or eukaryoticcells (such as those of animals, humans and higher plants).

[0038] Various libraries can be selected for large scale preparation.The construction of plasmid, cosmid, and phagemid cDNA libraries, orgenomic libraries are described in Sambrook, J. et al., In: MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)). Preferably, single-strandedphagemid cDNA libraries can be prepared as described previously byGruber, C. E. et al., Focus 15: (1993)). The general steps of the methodwill differ depending upon whether the desired sequence has been clonedinto single-stranded or double-stranded molecules, and whether suchmolecules are DNA or RNA.

[0039] As used herein, there is no constraint as to the sequence of thetarget nucleic acid molecule whose isolation is desired. Since thepresent invention relies upon nucleic acid hybridization, the targetmolecule should have a length of at least 10 nucleotides in order to beefficiently recovered. No upper limit to the size of the moleculesexists, and the methods of the invention can be used to isolate nucleicacid molecules of several kilobases or more.

[0040] The selection method of the present invention is based in partupon the observation that double-stranded nucleic acid moleculestransform bacterial cells with greater efficiency than single-strandednucleic acid molecules. In one embodiment, the invention achieves theisolation of a desired nucleic acid sequence from a library of sequencesby providing a primer molecule to the mixture. A “primer” or “primermolecule” as used herein is a single-stranded oligonucleotide or asingle-stranded polynucleotide that can be extended by the covalentaddition of nucleotide monomers during the template-dependentpolymerization reaction catalyzed by a polymerase. A primer is typically11 bases or longer; most preferably, a primer is 17 bases or longer.Examples of suitable DNA polymerases include the large proteolyticfragment of the DNA polymerase I of the bacterium E. coli, commonlyknown as “Klenow” polymerase, E. coli DNA polymerase I, thebacteriophage T7 DNA polymerase. Preferably, a thermostable polymerasewill be used, such as a polymerase that can catalyze nucleotide additionat temperatures of between about 50° C. to about 100° C. Exemplarythermostable polymerases are described in European Patent Appln.0258017, incorporated herein by reference. The thermostable “Taq” DNApolymerase (Life Technologies, Inc., Gaithersburg, Md.) is particularlypreferred.

[0041] Where the target mixture involved RNA molecules, and a DNAmolecule is desired, a reverse transcriptase may be employed. Reversetranscriptases are discussed by Sambrook, J. et al. (In: MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)) and by Noonan, K. F. et al. (Nucleic AcidsRes. 16:10366 (1988)). Similarly, where the target mixture involved RNA,an RNA polymerase may be used. Examples of suitable RNA polymerasesinclude E. coli RNA polymerase, T7 RNA polymerase, etc.

[0042] As a consequence of such polymerization, the desired targetmolecule, but not other nucleic acid molecules of the mixture, isconverted into a double-stranded form. The mixture can, without furtherprocessing, be transformed into suitable recipient bacteria (see,Sambrook, J. et al., In: Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).Transformants can be recovered, and their recombinant DNA or RNAmolecules can be extracted and retrieved. Such processing provides a newmixture or library of nucleic acid molecules that is substantiallyenriched for the desired molecule. Optionally, the above-describedmethod can be repeated (as often as desired) in order to obtain mixturesor libraries that are more highly enriched for the desired nucleic acidsequence.

[0043] A preferred method for conducting such processing employs alibrary or mixture of a single-stranded phagemid, such as M13. In such amethod, a primer is used to convert the single-stranded DNA moleculeinto a double-stranded form.

[0044] A. Capture Enrichment of Desired Molecules

[0045] In a first preferred embodiment, the selection method of thepresent invention can be augmented through the use of an optionalnucleic acid “capture” step. This embodiment is preferably performedusing single-stranded nucleic acid molecules. Where double-strandedcircular molecules are employed, a preferred initial step involvesdenaturing the molecules into their respective single strands. Mostpreferably, this is accomplished by transient incubation of the sampleat elevated temperatures (60-80° C. or above the T_(m) of the mixture).Alternatively, salt or ionic conditions can be adjusted, or denaturationcan be accomplished via helicase activity. The strand-separation stepmay require a topoisomerase in order to permit full strand separation.Alternatively, the double-stranded plasmid or linear target DNA could benicked and the nicked strand removed by denaturation or digestion. Inaddition, double-stranded linear DNA could be denatured or one stranddigested. These methods would leave one strand of DNA intact for hybridselection.

[0046] In accordance with the invention, such a population ofsingle-stranded molecules is then incubated in the presence of anoligonucleotide probe under conditions sufficient to permit and promotesequence-specific nucleic acid hybridization. Hybridization may beconducted under conditions which either permit or minimize randomhybridization. As used herein, conditions which minimize randomhybridization are of such stringency that they permit hybridization onlyof sequences that exhibit complete complementarity. In contrast,conditions that permit random hybridization will enable molecules havingonly partial complementarity to stably hybridize with one another.Suitable conditions which either permit or minimize random nucleic acidhybridization are described by Maniatis, T., et al. (In: MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratories, coldSpring Harbor, N.Y. (1982)), by Haymes, B. D., et al. (In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, DC (1985),herein incorporated by reference), and similar texts.

[0047] The probe is a nucleic acid molecule, preferably DNA, preferablygreater than 8-12 nucleotides in length, and most preferably greaterthan 15-30 nucleotides in length, whose sequence is selected to becomplimentary to the sequence of a region of the target molecule that isto be isolated. The probe thus need not be, and most preferably will notbe equal in size to the target molecule that is to be recovered. Twosequences are said to be “complementary” to one another if they arecapable of hybridizing to one another to form a stable anti-parallel,double-stranded nucleic acid structure. Thus, the sequences need notexhibit precise complementarity, but need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure. Thus, departures from complete complementarity arepermissible, 80 long as such departures are not sufficient to completelypreclude hybridization to form a double-stranded structure.

[0048] In some embodiments, the sequence of the probe and/or the primermay be derived from amino acid sequence data. In these instances, theprobe and/or the primer may have a degenerate sequence. For instance, ifone had an amino acid motif (e.g. zinc fingers) that occurred in anumber of proteins encoded in a library, one could enrich for nucleicacids encoding proteins having that motif.

[0049] In a preferred sub-embodiment, the probe is “haptenylated.” Asused herein, a “haptenylated” probe is a nucleic acid molecule that hasbeen covalently bonded to a hapten molecule. A hapten is a molecule thatcan be recognized and bound by another molecule, e.g. an antibody.Examples of haptens include any antigen, biotin, dinitrophenol, etc.Biotin is a preferred hapten of the present invention and may be boundby proteins such as avidin and streptavidin. The probe may be“haptenylated” using any of a variety of methods well known in the art.For example, methods for “biotinylating” the probe are described, forexample, by Hevey et al. (U.S. Pat. No. 4,228,237); Kourilsky et al.(U.S. Pat. No. 4,581,333); Hofman et al. (J. Amer. Chem. Soc.100:3585-3590 (1978)); Holmstrom, K. et al. (Anal. Biochem. 209:278-283(1993)), etc. Such modification is most preferably accomplished byincorporating biotinylated nucleotides into a nucleic acid moleculeusing conventional methods. Alternatively, such modification can be madeusing photobiotin (Vector Laboratories). Other methods can, of course,be employed to produce such biotinylated molecules.

[0050] The above-described incubation thus results in the hybridizationof the biotinylated probe and the desired target sequence such that adouble-stranded region is formed. In the next step of the preferredmethod, this complex is “captured” using a an immobilizable support,most preferably, a paramagnetic bead. In a preferred sub-embodiment, thecapture of the hybridized biotinylated probe is initiated without thenecessity for removing non-hybridized molecules.

[0051] The support is modified so as to have a binding ligand of biotinconjugated to it. Suitable binding ligands include avidin, orstreptavidin, or antibody or antibody fragments that bind biotin.Methods for effecting such covalent attachment are described by Hevey etal. (U.S. Pat. No. 4,228,237) and by Kourilsky et al. (U.S. Pat. No.4,581,333). Suitable solid supports include, but are not limited to,beads, tubes, or plates, which may be made of materials including, butnot limited to, latex, glass, polystyrene, polypropylene or otherplastic. Such supports can be 2-dimensional strips, beads, etc. Apreferred support is a paramagnetic-streptavidin conjugated bead, suchas the Dynabead Streptavidin M-280 bead (Dynal, Great Neck, N.Y.).

[0052] The addition of the beads (or other support) to the reactionpermits the biotinylated probe to bind to the avidin (or other biotinligand) of the beads. Such binding reactions are very strong. Forexample, the binding constant for the reaction between avidin and biotinis approximately 10¹⁵ 1/mole. The very strong nature of this bond hasbeen found to persist even when biotin is conjugated, by means of itscarboxyl group, to another molecule, or when avidin is attached toanother molecule.

[0053] As a consequence of such binding, any biotinylated probe that hashybridized to a desired target molecule will become bound to the bead orsupport. In contrast, non-target molecules will remain unbound, and canbe separated from the bound material by washing, filtration,centrifugation, sieving, or by magnetic separation methods. Mostpreferably, however, a magnet is used to pull the paramagnetic bead outof solution, and the beads are washed with a suitable buffer (such asone containing Tris, EDTA, and NaCl). Such treatment removes themajority of non-target nucleic acid sequences that were originallypresent, and hence eliminates undesired non-selected single-strandedphagemid DNA from the reaction.

[0054] The specifically captured single-stranded phagemid target DNA(hybridized to the biotinylated probe) is then released from the probeby treatment such as addition of an alkaline buffer, heat, etc. In apreferred sub-embodiment, the selected target DNA is resuspended in aformamide-Tris-EDTA buffer and released from the beads by heating at atemperature of 60-70° C. for a short period of time. The releasingtreatment is preferably selected such that the biotinylated proberemains attached to the magnetic beads, which is then removed fromsolution by sieving, centrifugation, filtration, or more preferably, bya magnet.

[0055] B. Nuclease Enrichment of Desired Molecules

[0056] In a second preferred embodiment, a nuclease enrichment protocolcan optionally be used to aid, or further aid in effecting the isolationof a desired target DNA molecule. Where employed, the second embodimentcan either be used alone, or in conjunction with the above-describedfirst preferred embodiment.

[0057] As in the case of the first preferred embodiment, single-strandednucleic acid is desired. Thus, where double-stranded nucleic acid isemployed, any of the above-described methods can be used to obtain asuitable single-stranded molecule. Such single-stranded molecules areconverted to their double-stranded DNA form using a DNA polymerase andin the presence of requisite nucleotide triphosphates, and factors, andan oligonucleotide primer. Where the first and second embodiments are tobe performed in conjunction, the primer employed in the secondembodiment may comprise all or a subset of the sequence present in thehaptenylated probe molecule of the first embodiment. In such asub-embodiment, therefore, the conversion step need not further sort ordistinguish among the recovered molecules. Preferably, stringenthybridization conditions are used and the conversion step is done athigh temperature with a thermostable polymerase, e.g. Taq polymerase. Inthis case, because the hybridization and conversion are done underconditions favorable to correct hybrids, the conversion step doesfurther enrich or select for the desired target molecule. In analternative sub-embodiment, the primer molecule may comprise a sequencethat although present on the desired target molecule was not containedon the “haptenylated” probe. Such a sub-embodiment permits theexperimentalist to purify only a subset of the initially presentmolecules. For example, if the probe was designed to hybridize to anymember of a gene library that had a particular enhancer sequence, andthe primer were designed to hybridize only to a receptor binding site,the net effect of the reaction would be to obtain double-strandedmolecules that contained both the enhancer sequence and the receptorbinding site.

[0058] Note that the hapten need not be covalently coupled to theprobe-nucleic acid. The hapten may be linked, either covalently ornon-covalently, to a molecule that non-covalently binds the probemolecule, e.g. a single-stranded DNA binding protein. The bindingprotein must bind tightly enough that significant quantities of it willnot fall off of the probe molecules and bind to nucleic acid moleculesof the sample.

[0059] The template-dependent extension of the primer is conducted inthe presence of at least “nucleotide analog” (either in lieu of or inaddition to the naturally occurring analog). A “nucleotide analog,” asused herein, refers to a nucleotide which is not found in the target DNAor RNA that is the primer's template. For example, where the isolatedtarget molecule is DNA, suitable nucleotide analogs includeribonucleotides, deoxyuridine, bromodeoxyuridine, 7-methylguanine,5-methyldeoxcytosine, 5,6-dihyro-5,6-dihydroxydeoxythymidine,3-methyldeoxyadenosine, etc. (see, Duncan, B. K., The EnzymesXIV:565-586 (1981)). Other nucleotide analogs will be evident to thosein the art. Where the template is RNA, deoxynucleotide triphosphates andtheir analogs are the preferred nucleotide analogs. Any single-strandednon-target DNA that remains after the conversion step may contribute abackground of non-target sequences in molecules recovered by the presentmethod, therefore, it is desirable to remove or eliminate anysingle-stranded DNA that might remain.

[0060] The presence of the nucleotide analog in the reaction will resultin the production of a double-stranded molecule that containsincorporated analog bases. Such incorporation affects the ability ofendonucleases and exonucleases to cleave or degrade the double-strandedmolecule. Thus, in a particularly preferred sub-embodiment, a primer isextended from a circular DNA template in the presence of a methylatednucleotide (for example, 5-methyl dCTP). The resulting double-strandedmolecule, which contains incorporated 5-methyl C residues, is resistantto cleavage by many restriction endonucleases. HhaI is particularlypreferred when used in conjunction with 5-methyl C, since it alsodegrades single-stranded DNA, the effect of incubation in the presenceof such enzymes is to destroy most or all residual undesired non-targetmolecules present, and to thereby greatly enrich the concentration ofthe desired vector. Other nucleotide analogs that inhibit or blockexonucleases or restriction endonucleases are 6-methyladenine,5-methylguanine and 5-methylcytidine. Combinations of nucleotide analogsand suitable enzymes are known in the art (see, for example, LifeTechnologies™ 1993-1994 Catalogue and Reference Guide, Chapter 6, LifeTechnologies, Inc., Gaithersburg, Md., herein incorporated byreference).

[0061] In a similar manner, where the source library was composed ofsingle-stranded RNA vectors, the use of dNTPs (i.e. dATP, dTTP, dCTP,and dGTP) in the conversion step will render such molecules resistant tomung bean nuclease, or Bal-31 nuclease.

[0062] Although the foregoing discussion has emphasized the use ofcircular molecules, the methods of the present invention are fullyamenable to the use of linear molecules. In such a case, the primermolecule (but not necessarily the probe molecule) is preferably selectedsuch that it hybridizes to the 5′ terminus of the target molecule. Suchselection will permit the template dependent extension of the moleculeto produce a full length copy of the target molecule.

[0063] Desirably, the recovered target DNA is then precipitated withorganic solvents, and resuspended in buffer. The product may then betransfected or electroporated into recipient cells, for example by themethod of Rubenstein et al. (Nucl. Acids Res. 18: 4833 (1990), hereinincorporated by reference).

[0064] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLE 1 Preferred Method for Isolating Desired Target Molecules

[0065] A preferred method for isolating a desired target moleculeemploys a library or mixture of a single-stranded phagemid, such as M13.In such a method, the single-stranded phagemid is introduced into an unqdut mutant of E. coli (Kunkel, T. A., U.S. Pat. No. 4,873,192; Longo, M.C. et al., Gene 93:125-128 (1990); Hartley, U.S. Pat. No. 5,035,966; allherein incorporated by reference). The “+” strand of phagemids grown insuch mutants contains deoxyuridine (dUTP), and can be recovered from thepackaged virion. Thus, the use of such mutants permits the isolation ofa library or mixture that comprises single-stranded DNA molecules whichcontain dU residues (Kunkel, T. A., U.S. Pat. No. 4,873,192).

[0066] The recovered DNA can then be optionally captured via a capturestep, or directly processed using a nuclease enrichment step.

[0067] If a capture step is to be conducted, the dU-containing strandsare incubated in the presence of a complementary biotinylated probe. Theprobe, and any hybridized DNA is then recovered by permitting the biotinto bind to avidin-coated paramagnetic beads, and then recovering thebeads from solution using a magnet. The library or mixture is recoveredfrom the beads by denaturation of the hybridized molecules.

[0068] The recovered single-stranded DNA is then incubated in thepresence of a complementary primer, dATP, dTTP, dCTP, and dGTP and underconditions sufficient to permit the extension of the primer. Suchextension thus creates a sample that contains single-strandeddU-containing molecules and double-stranded dU/dT hybrid (desiredtarget) molecules.

[0069] Although the triphosphate form of deoxyuridine, dUTP, is presentin living organisms as a metabolic intermediate, it is rarelyincorporated into DNA. When dUTP is incorporated into DNA, the resultingdeoxyuridine can be promptly removed in vivo by the enzyme uracil DNAglycosylase (UDG) (Kunkel, U.S. Pat. No. 4,873,192; Duncan, B. K., TheEnzymes XIV:565-586 (1981), both references herein incorporated byreference in their entirety).

[0070] In this embodiment of the present invention, the mixture ofmolecules is then treated, either in vivo or in vitro with UDG. Suchtreatment destroys all of the single-stranded, non-desired, non-targetmolecules in the sample. It further destroys the “+” strand of all ofthe double-stranded desired target molecules.

[0071] The sample is therefore then either directly transformed into E.coli to permit the isolation of the target molecule or incubated in thepresence of a primer molecule that is capable of hybridizing to the “−”strand of the phagemid. Such incubation is under conditions suitable formediating the template-dependent extension of the primer. Hence, suchincubation produces double-stranded molecules that have the sequence ofthe desired target molecules, and thereby permit the isolation of thetarget molecule.

EXAMPLE 2 Preparation of Single-stranded DNA

[0072] The large scale preparation of single-stranded phagemid cDNAlibrary was made as described previously (Gruber, C. E. et al., Focus 15(1993), herein incorporated by reference).

Preparation of Biotinylated Oligonucleotides

[0073] The oligonucleotide probes were biotin-labeled usingbiotin-14-dCTP and terminal deoxynucleotidyl transferase (TdT) asdescribed by Flickinger, J. L. et al. (Nucl. Acids Res. 20: 2382 (1992))with the following minor modifications. In a typical reaction, 0.3-0.5nmol (≈5 μg) of oligonucleotides (21-25-mer), 500 μM of biotin-14-dCTPand 60 units of TdT in 50 μl of 1× tailing buffer [100 mM potassiumcacodylate (pH 7.2), 2 mM CoCl₂ and 200 μM DTT] was incubated at 37° C.for 15 min. The reaction was terminated by adding 2 μl of 0.25 M EDTA.The labeled probes were precipitated by adding an equal volume (52 μl)of 1 M Tris buffer (pH 7.5), 10 μg glycogen as carrier, and 2.5 volume(260 μl) of ethanol, and stored on dry ice for 10 min. Aftercentrifugation at 4° C. for 10 min, the probes were rinsed with 100 μlof 75% ethanol and centrifuged for 2 min. The probes were air-dried anddissolved in 10 μl of TE. To determine the labeling efficiency and theconcentration of the labeled probe, 2 μl of labeled products wereresuspended in an equal volume of sequencing reaction stop buffer [95%(v/v) formamide, 10 mM EDTA (pH 8.0), 0.1% (w/v) bromophenol blue, 0.1%(w/v) xylene cyanol], heated at 95° C. for 1 min and chilled on ice. Theprobes were electrophoresed along with a known amount of the startingmaterial on 16% denaturing PAGE. The gel was soaked in a ethidiumbromide solution (0.5 μg/ml) for 15 min, and photographed. Typically,more than 95% of the oligonucleotide will be labeled. The concentrationof the labeled probes was determined by the comparison to the knownstarting material.

Hybrid Selection

[0074] The hybridization was performed by the following procedure: 1-10μg of single-stranded target library DNA was diluted with 10 μl ofdilution buffer (100 mM HEPES, pH 7.5, 2 mM EDTA and 0.2% SDS) to afinal volume of 19 μl in a 5 ml Falcon tube. The DNA was denatured at95° C. for 1 min and immediately chilled in ice water for 5 min. 1 μl(20 ng) of biotin-probe was added to the DNA mixture, followed by theaddition of 5 μl of 5 M NaCl. The hybridization mixture was incubated at42° C. with continuous shaking (200 rpm) in a culture incubator for 24h. Before binding the hybrids to the streptavidin, 50 μl of thestreptavidin coated paramagnetic beads (DYNAL) were washed once with 1×binding buffer (10 mM TRIS, pH 7.5, 1 mM EDTA and 1 M NaCl) by followingthe manufacturer's instructions. The paramagnetic beads were resuspendedin 20 μl of 1× binding buffer. The hybridization mixture was added tothe resuspended beads and mixed well. The mixture was incubated at roomtemperature for 1 h with occasional mixing by gently tipping the tube.The paramagnetic beads were separated from the DNA bulk by inserting thetube into the magnet, and washed 6 times with the washing buffer (10 mMTris, pH 7.5, 1 mM EDTA and 500 mM NaCl). Finally, the paramagneticbeads were resuspended in 20 μl of 30% formamide in TE buffer. Theselected DNA was released by heating the beads at 6520 C. for 5 min. Thetube was inserted into the magnet, and the aqueous phase was transferredto a new tube. The beads were washed once with 15 μl of TE buffer, andthe aqueous phases were pooled. The selected DNA was precipitated with0.5 volumes of 7.5 M ammonium acetate, 10 μg of glycogen, and 2.5 volumeof ethanol. The DNA pellet was dissolved in 5-10 μl of TE buffer. Analiquot (1 μl) was used for the electroporation to determine the hybridselection efficiency.

Repair of Single-Stranded DNA

[0075] The remainder of the selected single-stranded DNA was convertedto double-stranded DNA before electroporation as described by Rubensteinet al. (Nucl. Acids Res. 11: 4833 (1990)) with some modifications. Thereaction was carried out in 30 μl containing the selectedsingle-stranded DNA, 250 ng of unlabeled primer, 300 μM each dTTP, dGTP,dATP and 5-methyl dCTP, Taq DNA polymerase buffer and 2 units of Taq DNApolymerase. After repair, the mixture was extracted once withphenol:chloroform. The organic phase was back-extracted with 15 μl ofTE, the aqueous phases were pooled and ethanol precipitated. The pelletwas rinsed with 100 μl of 75% ethanol and dried. The repaired DNA wasdissolved in 5-10 μl of TE and digested with HhaI for 2 h at 37° C.After digestion, the mixture was extracted once with phenol:chloroform,ethanol precipitated and dissolved in 5-10 μl of TE.

Detection of the Target Gene

[0076] The repaired, digested DNA was used to transform E. coli bacteria(DH10B background) by chemical transformation or electroporation. Thetarget colony can be detected by the PCR, colony hybridization or cyclesequencing approach.

[0077] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A method for recovering a desired target nucleicacid molecule from a sample containing a mixture or library ofsingle-stranded nucleic acid containing said molecule, wherein saidmethod comprises the steps: A. incubating said sample containing saidnucleic acid mixture or library in the presence of a primer nucleic acidmolecule complementary to a sequence of said desired target molecule;said incubation being under conditions sufficient to permithybridization between said primer and said desired target molecule, andfurther sufficient to permit the template-dependent extension of saidprimer to thereby generate a double-stranded desired target molecule; B.transforming single-stranded and double-stranded members of said mixtureor library into a host cell, and C. recovering said desired moleculefrom said cell.
 2. The method of claim 1 , wherein prior to commencingstep A, said method comprises the presteps: (1) incubating an initialsample containing said nucleic acid mixture or library in the presenceof a haptenylated nucleic acid probe molecule, said probe moleculeshaving a sequence complimentary to a nucleotide sequence of said desiredtarget molecule; said incubation being under conditions sufficient topermit said probe to hybridize to said desired target molecule and tothereby generate a hybridized molecule wherein said target molecule isbound to said probe; (2) incubating said sample containing said nucleicacid mixture or library and biotinylated probe-target hybridizedmolecules of prestep (1) in the presence of a binding ligand of thehapten of said haptenylated probe, said binding ligand being conjugatedto support; said incubation being sufficient to permit said probemolecules, and said probe-target hybridized molecule to become bound tosaid binding ligand of said support; (3) recovering said probe-targethybridized molecules bound to said support from said nucleic acidmixture or library and any unbound biotinylated probe-target hybridizedmolecules of prestep (2); and (4) incubating said recovered supportcontaining said bound probe-target hybridized molecules under conditionssufficient to separate the strands of double-stranded molecules; saidincubation thereby releasing said hybridized target molecule from saidbiotinylated probe, and generating a sample single-stranded desiredtarget molecule for use in step (A).
 3. The method of claim 1 , whereinsaid single-stranded nucleic acid molecule of said sample contains anucleotide analog, and wherein after completing step A, but prior tocommencing step B, said method additionally comprises the presteps: (1′)incubating said generated double-stranded molecules in the presence of anuclease capable of degrading nucleic acid containing nucleotide analogresidues; and (2′) incubating non-degraded nucleic acid with a primerunder conditions sufficient to permit said primer to be extended in atemplate-dependent manner.
 4. The method of claim 1 , wherein in step A,said template-dependent extension of said primer is conducted in thepresence of a nuclease resistant nucleotide analog to thereby generate adouble-stranded desired target molecule containing a residue of saidnucleotide analog; and wherein prior to commencing said step B, saidmethod additionally comprises the presteps: (1″) incubating saidgenerated double-stranded desired target molecule in the presence of anuclease, wherein said nuclease is substantially unable to cleave anucleic acid molecule containing said nucleotide analog residue, but issubstantially capable of degrading both single-stranded nucleic acidmolecules and double-stranded nucleic acid molecules that lack saidnucleic acid analog residue; said incubation being under conditionssufficient to permit such degradation, and thereby substantiallyeliminating both single-stranded nucleic acid molecules anddouble-stranded nucleic acid molecules that lack said nucleic acidanalog residue from said sample; and thereby forming a preparationhaving a substantial enrichment of said desired target molecule relativeto said initial sample; and (2″) recovering said desired molecule fromsaid preparation of prestep (1″) to thereby form a library or mixturefor said step B.
 5. The method of claim 1 , wherein in step A saiddesired incubation is under conditions which minimize randomhybridization.
 6. The method of claim 2 , wherein in prestep (1) saiddesired incubation is under conditions which minimize randomhybridization.
 7. The method of claim 1 , wherein said desired targetnucleic acid molecule is a DNA molecule.
 8. The method of claim 7 ,wherein said DNA molecule is a single-stranded DNA molecule.
 9. Themethod of claim 1 , wherein said desired target nucleic acid molecule isan RNA molecule.
 10. The method of claim 1 , wherein said desired targetnucleic acid molecule is a single-stranded nucleic acid molecule. 11.The method of claim 1 , wherein said desired target molecule is acircular nucleic acid molecule.
 12. The method of claim 11 , whereinsaid desired target molecule is a circular DNA molecule.
 13. The methodof claim 2 , wherein said hapten is biotin, and wherein said bindingligand of said hapten is avidin, streptavidin, or an antibody orantibody fragment that binds biotin.
 14. The method of claim 13 ,wherein said/binding ligand of biotin is avidin.
 15. The method of claim13 , wherein said binding ligand of biotin is streptavidin.
 16. Themethod of claim 2 , wherein said support of said prestep (2) is aparamagnetic bead.
 17. The method of claim 16 , wherein saidhaptenylated probe-target hybridized molecule bound to said paramagneticbead is recovered by magnetic means.
 18. The method of claim 2 , whereinin said primer molecule of step A is complementary to the same sequenceof said desired target molecule as said probe molecule of substep (1).19. The method of claim 2 , wherein in said primer molecule of step A iscomplementary to a sequence of said desired target molecule that differsfrom the sequence of said desired target molecule that is complementaryto said probe molecule of substep (1).
 20. The method of claim 3 ,wherein said nucleic acid analog is deoxyuridine, and wherein saidnuclease is UDG.
 21. The method of claim 4 , wherein said nuclease doesnot cleave hemimethylated DNA.
 22. The method of claim 21 , wherein saidnucleic acid analog is 5-methylcytidine, and wherein said nuclease thatdoes not cleave hemimethylated DNA is HhaI.
 23. The method of claim 2 ,wherein in prestep (1), said probe has a degenerate sequence.
 24. Themethod of claim 4 , wherein in step A, said primer has a degeneratesequence.
 25. The method of claim 1 , wherein said host cell is abacterium.
 26. The method of claim 1 , wherein said method additionallyincludes the step of amplifying said desired target molecule by an invitro amplification reaction.
 27. The method of claim 26 , wherein saidin vitro amplification reaction is a polymerase chain reaction.